Yinong Yang1, Qing Li2, Feng Tan3, Jun Zhang1, Wu Zhu1. 1. Medical School, Hunan University of Medicine, Huaihua, Hunan 418000, P.R. China. 2. Nursing School, Hunan University of Medicine, Huaihua, Hunan 418000, P.R. China. 3. School of Stomatology, Hunan University of Medicine, Huaihua, Hunan 418000, P.R. China.
Acute lung injury (ALI) is a type of lung immunity that is produced by the human body in response to lung disease or other acute diseases. ALI is clinically characterized by dyspnea and can develop into acute respiratory distress syndrome (1,2). High vascular permeability, oxidative stress, and autoimmunity may all affect the occurrence and progress of ALI (3–5). Understanding the mechanism of ALI is beneficial for the development of ALI therapy.Inflammation of alveolar neutrophils is a sign of human ALI. Interleukin-8 (IL-8) is a neutrophil activator which can combine with auto-antibodies to form a complex and activate chemotaxis of neutrophils in the form of the complex, to achieve the purpose of delaying apoptosis of neutrophils (6–8). Such an inhibition of apoptosis of neutrophils aggravates the inflammatory response. Liu et al (9) have confirmed the close relationship between serum IL-8 concentration and the high risk of suffering from ALI. Therefore, IL-8 plays an indispensable role in ALI.Changes in the structure and function of surfactant proteins, such as surfactant protein A (SP-A) and surfactant protein B (SP-B), increase the susceptibility to lung diseases (10). SP-A not only maintains the epithelial integrity by inhibiting lung cell apoptosis, but also controls inflammatory response by inhibiting inflammatory cytokines, such as IL-8, TNF-α and IL-β (11,12). It can also combine with apoptotic neutrophils, enhancing the phagocytosis of macrophages on apoptotic neutrophils, and accelerating the clearance to apoptotic cells (13). Downregulation of SP-B causes changes in the surface active function and inflammatory cytokines, such as TNF-α, IL-β and IL-6, leading to pulmonary dysfunction (14). In addition, SP-B stimulates the exchange and transportation of calcium ions in alveoli by inducing cell autocrine or paracrine to maintain alveolar information transmission (15).The above indicate that there may be a certain connection between IL-8 and SP-A and SP-B, and this connection may have an impact on ALI. At present, there is no report on the relationship among the three factors and ALI. In the present study, in order to investigate whether IL-8 causes ALI through SP-A and SP-B, an ALI model for A549 cells was constructed, the changes of IL-8, SP-A and SP-B in this process were determined, and the relevant mechanism of action of the three in ALI was analyzed.
A total of 53 ALIpatients admitted to the Hunan University of Medicine Hospital (Huaihua, China), from January 2017 to March 2018, were selected as research subjects, including 32 males and 21 females, 52.39±3.21 years of age. The inclusion criteria were: Patients confirmed with ALI, and patients without past treatment history. The exclusion criteria were: Patients with comorbid malignant tumors, comorbid infectious diseases, or mental disease; and patients unwilling to cooperate with the treatment. Another 56 healthy subjects, who underwent physical examination, were enrolled as a control group, including 34 males and 22 females, 51.02±2.77 years of age. Patients and healthy subjects participating in this study were all informed of the study and had complete clinical data. The study was carried out under the permission of the Hospital Ethics Committees. The study was approved by the Ethics Committee of Hunan University of Medicine. Signed informed consents were obtained from the patients or their guardians.
Determination of serum IL-8, SP-A, and SP-B levels in ALI patients by ELISA
Fasting blood was collected from ALIpatients, and centrifuged at 1,000 × g under 4°C for 10 min to obtain the supernatant. The collected supernatant was stored in a refrigerator at −80°C for further analysis. The IL-8, SP-A and SP-B ELISA kits were used to determine the content of corresponding protein in serum during ELISA assay.
Inducing A549 cells by lipopolysaccharides (LSPs)
A549 cells were cultured at 37°C/5% CO2 animal cell incubator with cell culture medium containing DMEM + 10% fetal bovine serum + 1% penicillin/streptomycin (100X). Subsequent experiment was carried out after the cells were cultured to reach 80–90% confluency.
A total of 1 ml of pancreatin was added to the medium for cell enzymolysis
After 2 min, the enzyme solution was removed, and 2 ml of fresh culture medium was added. A pipette was used to gently blow the culture medium to prepare a cell suspension. Countess™ Automated Cell Counter was used to count the cells in the cell suspension. Cells were seeded into a 6-well plate at 2×106/well, and 2 ml of fresh culture medium were added to each well. LSP was used to treat A549 cells for 3 h with a concentration increasing gradient of 0.01–10 µg/ml). The incubator environment was maintained at 37°C and 5% CO2.
Determination of cell viability by MTT assay
Cells were subjected to enzymolysis for 2 min. Then, the enzyme solution was removed and fresh culture medium was added to prepare cell suspension. The cells were seeded into four 96-well plates, 5×103 cells/100 µl in each well, with 3 wells in each group. One plate was taken out every 24 h, 10 µl/well of 5 mg/ml MTT solution, solved in DMSO (Beijing Solarbio Science & Technology Co., Ltd.), were added and the cells were cultured continuously for 1 h. Subsequently, the medium was sucked out, and the optical density (OD) at 570 nm was measured using an enzyme mark instrument. The experiment was repeated 3 times, and a cell viability-time curve was drawn.
Determination of cell apoptosis by flow cytometry
Cell suspension with 1×106 cells was prepared. The cells were immobilized in 70% ice-cold ethanol solution, with ambient temperature controlled at 4°C. The ethanol solution was removed, and the cells were incubated in Annexin V-FITC/7-AAD mixed solution. Then the FACScan flow cytometer was employed to analyze apoptosis. The cells were analyzed by FACScan flow cytometer (Becton, Dickinson and Co.) and the data were analyzed using Phoenix Flow Systems (Innovative Cell Technologies, Inc.). The cells were incubated in Annexin V-FITC/7-AAD Apoptosis Detection Kit (Elabscience Biotechnology Co., Ltd.).
Determination of protein expression by western blot analysis
Protein extract (1 ml) was added into a culture dish, and a pipette was used to repeatedly blow the solution until the cells were fully pyrolyzed. The protein extraction buffer included 20 mM Tris-HCl (pH 7.5) and protease inhibitor (both from Beijing Solarbio Science & Technology Co., Ltd.). The solution was sucked out, placed in an Eppendorf tube, and centrifuged at 16,000 × g under 4°C for 15 min to collect the supernatant. The protein to be detected via the BCA method was separated using 15% polyacrylamide gel electrophoresis. Protein was transferred to a nitrocellulose (NC) membrane and was let to stand at room temperature for 1 h (blocked with 5% skim milk - phosphate-buffered saline (PBS) solution). The mass of protein loaded per lane was 15 µg. IL-8, SP-A, SP-B, caspase-9, caspase-3, Bax, Bcl2, TNF-α, IL-17 and IL-1β primary antibodies were added, and let to stand overnight at 4°C. The NC membrane was washed with PBS solution 3 times, and then HRP conjugated goat anti-rabbit secondary antibody was added, and let to stand for 1 h at room temperature. Finally, the NC membrane was washed with PBS solution, and visualized by the enhanced chemiluminescence method. ECL Western Blotting Substrate (Thermo-Fisher Scientific, Inc.) was used for visualization. The internal reference protein was β-actin and the relative expression level of the protein to be detected was calculated as (the gray value of the band to be detected)/(the gray value of β-actin protein band).
Verification of protein-protein interaction by co-immunoprecipitation (Co-IP)
Protein was extracted as described above. PBS was applied to wash the protein A/G agarose beads 2 times, and then used to prepare 50% protein A/G agarose bead solution. Subsequently, 50% protein A/G agarose bead solution was added into the protein samples to remove non-specific binding proteins. The samples were added with IL-8 antibody and 5 µg of SP-A or SP-B antibody, and slightly shaken overnight at 4°C. Then the samples were centrifuged at 1,000 × g for 5 min under 4°C, and the supernatant was discarded. The samples were washed 4 times with 1 ml of Co-IP buffer. Western blot analysis was employed to identify the precipitated protein as described above.
Statistical analysis
Data were statistically analyzed by SPSS 20.0 [Asia Analytics (formerly SPSS China)], and graphs were generated using GraphPad Prism 6 (GraphPad Software). Measurement data were expressed as the mean ± SD. Comparison between two groups was carried out by independent samples t-test, and comparison among multiple groups was carried out using one-way ANOVA. Post hoc pairwise comparison was performed by the LSD t-test. Pearson's correlation analysis was carried out to analyze the correlation of IL-8 with SP-A and SP-B. The data were analyzed using two-sided test. Ninety-five percent was used as the confidence interval. P<0.05 was considered to indicate a statistically significant difference.
Results
High expression of serum IL-8, and low expression of SPA and SPB in ALI patients
Serum was collected from 53 ALIpatients and 56 healthy subjects, and the IL-8, SP-A, and SP-B contents in the serum samples were determined using ELISA. Compared with healthy subjects, ALI patients showed significantly increased serum IL-8 level; however, significantly decreased serum SP-A and SP-B levels (all P<0.05). The results suggest that ALI is associated with high expression of IL-8 protein and low expression of SP-A/B protein (Fig. 1).
Figure 1.
Expression of serum IL-8, SP-A and SP-B in ALI patients. (A) IL-8 was highly expressed in the serum of ALI patients. (B) SP-A expression was low in the serum of ALI patients. (C) SP-B expression was low in the serum of ALI patients. ***P<0.001, compared with control group. IL-8, interleukin-8; SP-A, surfactant protein A; SP-B, surfactant protein B; ALI, acute lung injury.
Negative correlation of IL-8 with SP-A and SP-B in ALI patients
IL-8 was highly expressed in the serum of ALI patients, while SP-A and SP-B expression was low in the serum. Thus, Pearson's correlation analysis was employed to explore the correlation of IL-8 with SP-A and SP-B. As shown in Fig. 2, IL-8 was negatively correlated with SP-A and SP-B, respectively (r=−0.6421 and −0.7019, respectively; P<0.0001).
Figure 2.
Pearson's correlation analysis of IL-8 with SP-A and SP-B. IL-8 was negatively correlated with (A) SP-A and (B) SP-B. IL-8, interleukin-8; SP-A, surfactant protein A; SP-B, surfactant protein B.
Construction of ALI model by inducing A549 cells by LSPs
An ALI model was constructed by inducing normal A549 cells by LSP, and western blot analysis was carried out to determine the changes of IL-8, SP-A and SP-B levels in ALI model cells. The results are shown in Fig. 3. Compared with normal A549 cells, LSP-induced A549 cells showed significantly increased IL-8 level; however, significantly decreased SP-A and SP-B levels (all P<0.05).
Figure 3.
Construction of the ALI model by inducing A549 cells with LSPs. (A) IL-8 level in the ALI model cells was increased; ***P<0.001. (B) SP-A level in the ALI model cells was decreased; **P<0.01. (C) SP-B level in the ALI model cells was decreased; *P<0.05 and **P<0.01. ALI, acute lung injury; LSP, lipopolysaccharides; IL-8, interleukin-8; SP-A, surfactant protein A; SP-B, surfactant protein B.
Effects of IL-8 on ALI model cells
In order to study the biological functions of IL-8 in ALI model cells, siRNA was adopted to silence IL-8, and the western blot analysis was carried out to determine the expression of SP-A, SP-B, caspase-9, caspase-3, Bax, Bcl2, TNF-α, IL-17 and IL-1β. MTT assay was used to detect cell viability, and flow cytometry to detect apoptosis. The results are shown in Fig. 4. Compared with the NC si group, the IL-8 siRNA group showed increased SP-A and SP-B levels (Fig. 4A and B), intensified cell viability (Fig. 4C), decreased apoptosis rate (Fig. 4D), decreased levels of caspase-9, caspase-3, Bax, TNF-α, IL-17 and IL-1β, and increased Bcl2 level (Fig. 4E and F). The above results indicate that IL-8 promotes cell apoptosis, inhibits cell viability and expression of SP-A and SP-B, and aggravates cell inflammatory reaction.
Figure 4.
Effects of IL-8 on ALI model cells. (A) Inhibiting IL-8 increased SP-A expression; ***P<0.001. (B) Inhibiting IL-8 increased SP-B expression; ***P<0.001. (C) Inhibiting IL-8 increased cell viability; *P<0.05, compared with the NC si group. (D) Inhibiting IL-8 decreased the cell apoptosis rate; *P<0.05. (E) Inhibiting IL-8 downregulated the expression of caspase-9, caspase-3, and Bax, and upregulated the expression of Bcl2; *P<0.05, compared with the NC si group for the same protein. (F) Inhibiting IL-8 lowered the levels of TNF-α, IL-17, and IL-1β; *P<0.05, compared with the NC si group for the same cytokine. IL-8, interleukin-8; ALI, acute lung injury; SP-A, surfactant protein A; SP-B, surfactant protein B.
Effects of SP-A and SP-B on ALI model cells
In order to study the biological functions of SP-A and SP-B in ALI, SP-A and SP-B in ALI model cells were silenced, and western blot analysis was carried out to determine the expression of IL-8, caspase-9, caspase-3, Bax, Bcl2, TNF-α, IL-17 and IL-1β. MTT assay was used to detect cell viability, and flow cytometry to detect apoptosis. The results are shown in Fig. 5. Compared with the control group, the SP-A siRNA group, SP-B siRNA group and SP-A siRNA + SP-B siRNA group all showed increased expression of IL-8 (Fig. 5A), decreased cell viability (Fig. 5B), increased cell apoptosis rate (Fig. 5C), increased levels of caspase-9, caspase-3, Bax, TNF-α, IL-17 and IL-1β, decreased Bcl2 level (Fig. 5D and E), and the greatest difference occurred between the SP-A siRNA + SP-B siRNA group and the control group. The above results suggest that SP-A and SP-B inhibit cell apoptosis, enhance cell viability, decrease the expression of IL-8, TNF-α, IL-17 and IL-1β, and relieve cell inflammatory reaction.
Figure 5.
Effects of SP-A and SP-B on ALI model cells. (A) SP-A and SP-B inhibition of IL-8 expression; *P<0.05 and **P<0.01. (B) Silencing of SP-A and SP-B reduced cell viability; *P<0.05, compared with the control group. (C) SP-A and SP-B inhibition of apoptosis; **P<0.01 and ***P<0.001. (D) SP-A and SP-B inhibition of the expression of pro-apoptotic proteins caspase-9, caspase-3 and Bax, and promotion of the expression of pro-apoptotic protein Bcl2; *P<0.05 and **P<0.01, compared with the control group for the same protein. (E) SP-A and SP-B inhibition of the expression of TNF-α, IL-17 and IL-1β. *P<0.05, compared with the control group for the same cytokine. SP-A, surfactant protein A; SP-B, surfactant protein B; ALI, acute lung injury; IL-8, interleukin-8.
Verification of the correlation of IL-8 with SP-A and SP-B through Co-IP
In order to determine whether IL-8 can interact with SP-A and SP-B, respectively, the Co-IP method was employed. IL-8 antibody was used to precipitate IL-8 from total protein, and SP-A and SP-B antibodies were used as primary antibodies for western blot analysis to determine whether precipitated IL-8 protein contains SP-A and SP-B. Then, SP-A or SP-B antibody was used to precipitate SP-A or SP-B from total protein, and IL-8 antibody was used as primary antibody in western blot analysis to detect whether SP-A or SP-B had interaction with IL-8. The results are shown in Fig. 6. Fig. 6A shows that there were bands at 35 and 14 kDa in the input group, whereas the SP-A molecular weight was ~35 kDa and the SP-B molecular weight was ~14 kDa. Thus, the input group had SP-A and SP-B bands at 35 and 14 kDa, respectively. Similar bands appeared at 35 and 14 kDa in the IL-8 IP group; however, no band appeared at 35 and 14 kDa in the IgG IP group. The above positive precipitation results indicate that IL-8 could bind to SP-A and SP-B. The reverse precipitation results (Fig. 6B and C) showed that the input group, SP-A IP group and SP-B group had the same IL-8 band (molecular weight of ~9 kDa) at 9 kDa. Thus, it can be concluded that IL-8 can interact with SP-A and SP-B, respectively.
Figure 6.
Verification of the correlation of IL-8 with SP-A and SP-B through Co-IP. (A) IL-8 was positively precipitated, and the western blot was used to detect SP-A (35 kDa) and SP-B (14 kDa) in the precipitated protein. (B) SP-A was reversely precipitated, and the western blot was used to verify the presence of IL-8 (9 kDa) in the precipitated protein. (C) SP-B was reversely precipitated, and the western blot was used to verify the presence of IL-8 (9 kDa) in the precipitated protein. IL-8, interleukin-8; SP-A, surfactant protein A; SP-B, surfactant protein B; Co-IP, co-immunoprecipitation.
Discussion
Protein-protein interaction can affect the biological function diversity of numerous cell types, so it plays a very important role in the process of lung injury (16). Han et al (17) have reported that external mechanical force could be converted into biochemical signals through interaction between mucin-like protein and α-actin, interaction between c-Src and AFAP, or other protein-protein interactions, eventually damaging the lung cells. Interaction between Bcl-2 and Beclin-1 can inhibit apoptosis and autophagy (18), and α7nAchR protein of ALI cells can promote the interaction after being activated, thus playing a role in protecting cells and reducing apoptosis and autophagy (19). Hough et al (20) have confirmed that the activation of uncoupling protein-2 would increase vascular permeability, thus activating the calcineurin-cofilin-actin cascade reaction, disrupting protein-protein interaction related to endothelial barrier stability, leading to ALI. Zhang et al (21) have found that in lung injury, protein-protein interaction network mainly involves VEGF pathway, FoxO pathway, focal adhesion pathway and chemokine pathway. The results of this study revealed that IL-8 was highly expressed in ALI, whereas SP-A and SP-B expression was low, and IL-8 was negatively correlated with SP-A and SP-B, respectively. Therefore, it is speculated that IL-8 may play a role in ALI by interacting with SP-A and SP-B.In the present study, inhibiting IL-8 was shown to lead to obviously increased expression of SP-A and SP-B, downregulated expression of TNF-α, IL-17 and IL-1β, decreased expression of apoptosis proteins caspase-3, caspase-9 and Bax, increased cell viability and lowered apoptosis. The high expression of IL-8 in ALI has been confirmed previously (22). The above results suggest that the significance of IL-8 to ALI is not only in promoting the expression of pro-inflammatory factors including TNF-α, IL-17 and IL-1β to intensify inflammatory response, but also in inhibiting SP-A and SP-B, promoting cell viability, and suppressing apoptosis. Therefore, IL-8 exacerbates cell damage in ALI through the above mechanisms.In order to further study the specific mechanism of SP-A and SP-B in ALI, interference processing on SP-A and SP-B was also carried out. The results revealed that inhibited SP-A and SP-B caused decreased cell viability, increased apoptosis, and induced inflammatory reaction in cells. A number of studies have concluded that SP-A and SP-B can inhibit inflammatory reaction (23–25). In addition, SP-A also plays an important role in cell apoptosis (11). Therefore, based on the inhibitory effects on SP-A and SP-B, we consider that IL-8 can mediate the regulation by SP-A and SP-B on inflammation and apoptosis, eventually leading to ALI. The increase of intracellular calcium ions can induce passive depolymerization of cytoskeleton and destroy endothelial barrier (20), and SP-B has protective effects on calcium ion transport (15). Therefore, inhibition of SP-B by IL-8 may cause calciumhomeostasis disorder and ALI.The present study focused on exploring the interaction between IL-8 and SP-A and SP-B at the protein level to establish the effects of the interaction on ALI. Moreover, the results of this study suggest the potential value of IL-8, SP-A and SP-B in ALI treatment.Collectively, this study explored the roles of IL-8, SP-A, and SP-B in ALI by studying the interaction between IL-8 and the other two factors. IL-8 promotes cell apoptosis, inhibits cell activity and eventually cause ALI by inhibiting SP-A and SP-B protein levels, so inhibition of IL-8 or promotion of SP-A and SP-B levels may be the direction of ALI treatment.
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